Immunoglobulin (Ig) refers to the immunity-conferring portion of the globulin proteins of serum. The terms antibody and immunoglobulin are frequently used interchangeably. Ig's are secreted by differentiated B cells termed plasma cells; the Ig monomers are made up of two identical heavy chains (H) and two identical light chains (L). Each light chain is attached to a heavy chain by disulfide bonds, and the heavy chains are also attached to each other by one or more disulfide bonds. Each chain is a sequence of amino acids folded into a series of globular domains, the four chains together form a "Y"-shaped structure.
Each antibody or immunoglobulin has specific binding affinity for the foreign material or antigen that stimulated its synthesis. The globular domains at the branched ends of the "Y"-shaped molecule, termed variable domains (V), bind antigen. The variable domains together form a binding cavity that is geometrically and chemically complementary to a single type of antigen, allowing a fit of the cavity with the antigen. These domains are referred to as "variable" because the amino acid sequence of these domains varies according to antibody specificity. For example, an antibody binding the protein insulin will have a different amino acid sequence at the "variable" domains (binding sites) compared to an antibody that binds the protein keratin.
The globular domains of the "stem" portion of the "Y"-shaped molecule are called constant (C) domains. The amino acid sequences of these constant domains, for the most part, do not vary. However, sufficient differences exist for five major classes of Ig's to be identified in serum, each class containing a specific heavy chain. These "constant" portions of an immunoglobulin molecule determine biological activity such as the ability of immunoglobulins to bind to cells, to fix complement, and to traverse the placenta, for example. These classes of immunoglobulins and their heavy chains are as follows:
IgG (.gamma. chain): the principal immunoglobulin in serum, the main antibody raised in response to an antigen, this antibody crosses the placenta; PA1 IgE (.epsilon. chain): this Ig binds tightly to mast cells and basophils, and when additionally bound to antigen, causes release of histamine and other mediators of immediate hypersensitivity; plays a primary role in allergic reactions, hay fever, asthma, and anaphylaxis; this role does not appear to serve any useful purpose for a human; may serve a protective role against parasites; PA1 IgA (.alpha. chain): the Ig present in external secretions, such as saliva, tears, mucous, and colostrum; PA1 IgM (.mu. chain): the Ig first induced in response to an antigen, it has lower affinity than antibodies produced later and is pentameric; and PA1 IgD (.delta. chain): this Ig is found in relatively high concentrations in umbilical cord blood, may be an early cell receptor for antigen, and is the main lymphocyte cell surface molecule.
An IgG, IgE, IgA, IgM, and IgD antibody may have the same variable regions, i.e., the same antigen binding cavities, even though they differ in the constant region of their heavy chains.
These classes of immunoglobulins are also described as being isotypes of immunoglobulins. Further, immunoglobulin isotypes have subclasses, for example, IgG has four subclasses of heavy chains, .gamma.1, .gamma.2, .gamma.3, and .gamma.4, each different in structure and biological properties; IgE has one heavy chain, .epsilon.; IgA has two subclasses of heavy chain, .alpha.1 and .alpha.2; IgM has one heavy chain .mu.; and IgD has one heavy chain .delta.. A light chain of an immunoglobulin is either a kappa or lambda chain.
During development, stem cells formed in a yolk sac, liver, or bone marrow migrate to lymph nodes and the spleen where individual cell lines undergo clonal development independent of antigen stimulation. Most cells initially produce IgM, and later switch to IgG, IgE, or IgA production. Once B cells are released into the circulation and reach peripheral lymphoid tissues, they are capable, if stimulated by antigen, of differentiating into plasma cells that produce antibody specific for the antigen encountered.
Antibody diversity is a consequence of somatic recombination in germline DNA-encoding immunoglobulin regions, and RNA splicing of transcripts. Germline DNA carries instructions for nine constant region genes encoding the heavy chain isotype subclasses in linear order; .mu., .delta., .gamma.3, .gamma.1, .alpha.1, .gamma.2, .gamma.4, .epsilon., and .alpha.2. Isotype switching, DNA deletion, transcription and splicing have been the proposed mechanisms for producing the final messenger RNA that is translated by ribosomes to form a heavy chain immunoglobulin protein.
Ig isotype switching is preceded by germline transcription from the heavy chain locus that will subsequently undergo switch recombination. Germline transcription initiates at a non-coding exon (termed I exon) located upstream of a switch region; I exons generally display multiple stop codons in all reading frames and cannot code peptides of significant length (2-7). Germline transcription has been thought to participate in class switching by altering chromosome structure to provide recombinase accessibility (5-7). Although spliced and processed germline transcripts have been shown to be necessary (8), the exact role of processed germline transcripts in isotype switching has remained heretofore unknown.
Shimizu et al. reportedly indicated that mice transgenic with a rearranged membrane human VDJ-C.mu. gene produced chimeric Ig mRNAs with human VDJ correctly spliced to endogenous mouse C.gamma.1 and C.epsilon. through a trans-splicing mechanism (14-17). Although trans-splicing was not demonstrated, a mouse hybrid germline transcript of I.mu.-C.gamma.2b was reportedly provided by Li et al. (43).
Trans-splicing has been well delineated in trypanosomes, where many mRNAs trans-splice using an identical 5'35 nucleotide leader (termed splicing leader) (4648). A similar splicing leader (22 nucleotides) has been identified in nemotodes (49). However, both of these organisms employ a unique catalytic intron for trans-splicing not known to be present in humans.
An IgE-mediated allergy reaction results from the binding of an allergen (such as found in pollen, dander or dust) to IgE that is bound to the surface of basophils and mast cells; such binding causes crosslinking of underlying receptors, and the subsequent release of pharmacological mediators, such as histamine, causes the well known symptoms of allergy. Common allergies are estimated to affect routinely 10 to 20% of the population.
Treatment of allergy is complex and variable, but can be divided into three main approaches. Environmental controls are designed to eliminate or at least minimize exposure to the allergen. Symptomatic drug therapy is required in the control of most common allergies. The many drugs used for this purpose include the antihistamines and systemic and topical corticosteroids and sympathomimetics. Immunotherapy of allergy is accomplished by administration of gradually increasing doses of allergen over a period of months or years with the hope that the patient will develop increasing tolerance to the allergen. The precise mechanism of immunotherapy still is unknown, however, clinical improvement in some patients correlates well with the level of IgG-blocking antibodies, which presumably act by binding the allergen and preventing its interaction with mast cell-bound IgE. Immunotherapy isn't consistently effective for all sufferers of allergic symptoms; further, the immunotherapy regimen can be costly and require significant discipline on the part of the patient for success. Risks include local reactions at the injection site, and the possibility of serious generalized allergic reactions. Treatment failures may result from improper selection of allergens, development of new sensitivities, improper use of environmental controls and problems associated with the allergenic extracts. Alternative treatment includes steroid injections that generally suppress the whole immune system, a strategy that, by definition, puts the patient at risk.
Prior to the present invention, there have been no compositions or methods available for direct stimulation or inhibition of synthesis by modulating switching at a nucleic acid level of a particular human immunoglobulin isotype. Therefore, the present inventors have searched for improvements in this art and provide the compositions and methods described herein.